Licorice Cream Promotes Full-Thickness Wound Healing in Guinea Pigs
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Research Article www.marmarapharmj.com Licorice cream promotes full-thickness wound healing in Guinea pigs Nafise HANAFI 1, 2, Fereshteh TALEBPOUR AMIRI 1 *, Somayeh SHAHANI 3, Reza ENAYATIFARD 4, Maryam GHASEMI 5, Abbas Ali KARIMPOUR 1 1 Department of Anatomy, Faculty of Medicine, Molecular and Cell Biology Research Center, Mazandaran University of Medical Sciences, Sari, Iran. 2 Student Research Committee, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran. 3 Department of Pharmacognosy and Biotechnology, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran. 4 Department of Pharmaceutics, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran. 5 Department of Pathology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran. * Corresponding Author. E-mail: [email protected] (F.T.A.); Tel. +981133543088; ORCID No: 0000-0002-4236- 7439 Received: 06 December 2017 / Revised: 13 February 2018 / Accepted: 18 February 2018 ABSTRACT: Impaired wound healing may cause economic and social problems and seriously reduce the quality of life. The uses of herbal medicines as new alternative treatments are under investigation. This study investigated the effect of Glycyrrhiza glabra L. extract on the full-thickness wound healing in Guinea Pig model. Eight male Guinea Pigs (700-800 g) were used to be creared a square full-thickness wound with 1.5 ×1.5 dimensions on the lumbodorsal area. The wounds were randomly divided into four groups: I; control, II; 1% phenytoin, III; 5% G. glabra and IV; 10 % G.glabra. On day 3, 5, 7, 9 and 12, wound size was measured for assessment of the percent of wound healing. In order to determine the wound healing activity, excisional biopsies were evaluated histopathologically on the 12th days of treatments. Acute inflammation, granulation tissue fibroblast maturation, collagen deposition, epidermal layer formation, neovascularization, keratin layer formation were evaluated according to the Abramov score method. Hydroalcoholic extract of G.glabra exhibited total phenolic and flavonoid contents of 114.1 ± 5.45 and 82.85 ± 6.38 mg, respectively. G.glabra creams (5% and 10% w/w)were significantly increased the epidermal formation, collagen deposition and neovascularization, and was decreased acute inflammation in comparison to the control group. Wound healing rate were increased in the G. glabra groups. G.glabra creams 10 % was more effective than 5% w/w. Our findings proved that 5% and 10% w/w G. glabra creams were effective in acute dermal wound healing. More studies with different doses of G. glabra extract are recommended. KEYWORDS: Glycyrrhiza glabra L.; dermal wound healing; flavonoid, phenol; Guinea pigs models. 1. INTRODUCTION Wound healing, a physiological process, is repair of damaged cells and tissues that occurs with an accurate and properly process. Phases of wound healing includes: hemostasis, inflammation, proliferation, and remodeling, which that overlap with each other [1]. Loss of skin integrity by trauma leads to an imbalance of hemostasis and so, wound healing process become impaired [2]. Dermal wounds are a major concern and seriously reduce the quality of life for patients. As well, dermal wounds, with high cost and an inert treatment is a public health problem [3]. So, trying to quickly close the cutaneous wound with functional and aesthetic results would be ideal goal of treatment [4]. On the other hand, impaired wound healing is the main source of mortality and morbidity, that is associated with high costs [5]. Natural products are safe, less side effect, cultured acceptability and with having excellent physiological properties are the source of new treatments for cutaneous wound healing [6]. Herbal drugs are inexpensive compared with synthetic drugs. So some researchers have focused on the potential healing properties of herbs and there are various reports on using herbal drugs in healing of skin injuries [7, 8]. Glycyrrhiza glabra L. (G. glabra, Licorice), a native of south-east Europe and south-west Asia , is one of the most widely used as a herbal medicine, natural sweetener, as an additive for flavoring and sweetening How to cite this article: Hanafi N, Talebpour Amiri F, Shahani S, Enayatifard R, Ghasemi M, Karimpour AA. Licorice cream promotes full-thickness wound healing in Guinea pigs. Marmara Pharm J. 2018; 22 (3): 411-421. © 2018 Marmara University Press https://doi.org/10.12991/jrp.2018.81 ISSN: 1309-0801 411 Hanafi et al. Marmara Pharmaceutical Journal Glycyrrhiza glabra L. and dermal wound healing Research Article tobacco, candies, and beverages [9] and skin-cosmetics [10]. Pharmaceutical therapeutic properties, such as antifungal [11] and antimicrobial [12] efficacy, antiviral and antitumoral activity [13], anti-bacterial [14], antioxidant [15], antiulcer [16] have been reported in the numerous articles, that are only some of possible therapeutic properties. The main biologically active components of licorice include: triterpene saponins, flavonoids, isoflavonoids, chalcones and glycyrrhizic acid which are responsible for the observed activities [17, 18]. G. glabra is a medicinal plant that abundantly used in ancient times for treat bacterial and fungal infections but so far only one study reported the wound healing activity of this plant on full-thickness dermal wound in rat model [19]. Studies have shown that G. glabra can have a role in wound healing of gastric [20], oral [21] and colitis [16] mucosal ulcers. Also, the extract of this plant was effective on burning wound healing [22]. Since pig skin is similar to human skin physiologically and anatomically [23], and based on above facts about the G. glabra properties, this study was conducted to evaluate the effects of G. glabra on full thickness wound healing in guinea pigs. 2. RESULTS 2.1. Total phenolic and total flavonoid contents The total phenolic and total flavonoid contents of the extract were 114.1 ± 5.45 mg gallic acid equivalents and 82.85 ± 6.38 mg quercetin equivalents per gram dried extract respectively. 2.2. Macroscopic observation of wound healing All animals studied survived in the surgical procedures and during study period without any complications. Symptoms of secondary infection were not observed in any wounds. Wound surface in the phenytoin and G. glabra groups were moist. In macroscopic observation, necrotic tissue covers most of the surface of the wound in control group and clearly became visible on the wound areas from the early days in the control group, while necrotic tissue was not seen on wounds in the treated groups. New epithelium was totally obvious at the edges of the wound with a pinkish color in the treatment (phenytoin and G. glabra) groups (Figure 1). Figure 1: Macroscopic morphology of the wound healing in groups after 12- day treatment by application of G. glabra cream. A) control, B) phenytoin, C) 5% and D) 10% G. glabra cream. The ratios of wound areas to the initial wound area (WHR%) were greater in the treatment group compared with the control group. 2.3. Wound healing rate findings Figure 2 shows the wound healing rate (WHR) in the groups. The ratios of wound areas to the initial wound area on days 3, 5, 7, 9 and 12 were calculated. In the 5% G. glabra group, the wound area decreased and scare of wounds healed on day 12, being smaller in wound area than other groups. Difference between groups was statistically significant. As shown in Table 1, until the 5th day, the average of WHR in the groups was seen a similar increase. The mean WHR in wound area were presented in figure 2. Consider the following graph: https://doi.org/10.12991/jrp.2018.81 Marmara Pharm J 2018; 22(3): 411-421 412 Hanafi et al. Marmara Pharmaceutical Journal Glycyrrhiza glabra L. and dermal wound healing Research Article Figure 2: The ratios of wound areas to initial area on days 3, 5, 7, 9 and 12 were drawn on a line graphs. There were significant differences between the treatment groups with control groups. In all groups, the WHR increased gradually until day 5 during the inflammatory phase and the proliferative phase, increased with more speed until day 12 in the phenytoin and G. glabra groups during the remodeling phase and it was a gradual increase in the control groups (P<0.05). As was shown in Table 1, there was significant difference in WHR between G. glabra groups with control group regarding the wound healing score determined by microscopic analysis of wounds. The most important changes that can be seen from the image are the formation of epidermis after G. glabra cream treatment compared to control group. G. glabra stimulated epithelialization. New epidermis in 5 and 10% G. glabra covered 62.02 and 64.5% respectively, of the wound surface on day 12, while in the control and phenytoin groups, new epithelium covered 39.86 and 54.89 % of the surface of the wound, and these differences were statistically significant in G. glabra groups compared with control group. G. glabra stimulated epithelialization, and seemed that is not dose-dependent. Analysis of WHR is showed in the table 1. Table 1. WHR during days 3, 5, 7, 9 and 12 after the wound creation. WHR/Groups 3th day 5th day 7th day 9th day 12th day Mean±SD Mean±SD Mean±SD Mean±SD Mean±SD Control 5.7±2.55 13.84±5.61 21.15±8.23 35.44±9.56 39.86±8.27 Phenytoin 8.84±2.26a 14.15±1.94 30.37±7.75 41.24±10.23 54.89±9.85 a 5% cream 8.41±1.6 12.83±1.51 31.52±11.49 51.9±9.91a 62.02±9.74 a 10% cream 8.54±1.23a 13.68±1.4 30.3±3.73 a 45.86±6.23 64.5±11.83 a Control; without any prescription, Phenytoin; wounds treated with 1% phenytoin, 5% cream; wounds treated with 5% G.